Homology model of human corticosteroid binding globulin: a study of its steroid binding ability and a plausible mechanism of steroid hormone release at the site of inflammation

Corticosteroid binding globulin (CBG) and thyroxin binding globulin (TBG) both belong to the same SERPIN superfamily of serine-proteinase inhibitors but in the course of evolution CBG has adapted to its new role as a transport agent of insoluble hormones. CBG binds corticosteroids in plasma, delivering them to sites of inflammation to modify the inflammatory response. CBG is an effective drug carrier for genetic manipulation, and hence there is immense biological interest in the location of the hormone binding site. The crystal structure of human CBG (hCBG) has not been determined, but sequence alignment with other SERPINs suggests that it conforms as a whole to the tertiary structure shared by the superfamily. Human CBG shares 52.15% and 55.50% sequence similarity with alpha1-antitrypsin and alpha1-antichymotrypsin, respectively. Multiple sequence alignment among the three sequences shows 73 conserved regions. The molecular structures of alpha1-antitrypsin and alpha1-antichymotrypsin, the archetype of the SERPIN superfamily, obtained by X-ray diffraction methods are used to develop a homology model of hCBG. Energy minimization was applied to the model to refine the structure further. The homology model of hCBG contains 371 residues (His13 to Val383 ). The secondary structure comprises 11 helices, 15 turns and 11 sheets. The putative corticosteroid binding region is found to exist in a pocket between beta-sheets S4, S10, S11 and alpha helix H10. Both cortisol and aldosterone are docked to the elongated hydrophobic ligand binding pocket with the polar residues at the two extremities. A difference accessible surface area (DASA) study revealed that cortisol binds with the native hCBG more tightly than aldosterone. Cleavage at the Val379-Met380 peptide bond causes a deformation of hCBG (also revealed through a DASA study). This deformation could probably trigger the release of the bound hormone. Figure Stereoscopic view of the ribbon diagram of hCBG complexed with cortisol. The bound cortisol is shown in space filling model in blue. Helices and sheets are shown in red and magenta respectively. Turns are shown in yellow.     

Unusual susceptibility of heme proteins to damage by glucose during non-enzymatic glycation

Glucose modifies the amino groups of proteins by a process of non-enzymatic glycation, leading to potentially deleterious effects on structure and function that have been implicated in the pathogenesis of diabetic complications. These changes are extremely complex and occur very slowly. We demonstrate here that hemoglobin and myoglobin are extremely susceptible to damage by glucose in vitro through a process that leads to complete destruction of the essential heme group. This process appears in addition to the expected formation of so-called advanced glycation end products (AGEs) on lysine and other side-chains. AGE formation is enhanced by the iron released. In contrast, the heme group is not destroyed during glycation of cytochrome c, where the sixth coordination position of the heme iron is not accessible to solvent ligands. Glycation leads to reduction of ferricytochrome c in this case. Since hydrogen peroxide is known to destroy heme, and the destruction observed during glycation of hemoglobin and myoglobin is sensitive to catalase, we propose that the degradation process is initiated by hydrogen peroxide formation. Damage may then occur through reaction with superoxide generated (a reductant of ferricytochrome c), or hydroxyl radicals, or with both.     

Effect of solvent residues of Vitex negundo Linn. and Cassia fistula Linn. on pulse beetle, Callosobruchus maculatus Fab. and its larval parasitoid, Dinarmus vagabundus (Timberlake)

Effect of solvent residues of Vitex negundo L. and Cassia fistula L. leaves (0.5 and 1%) was studied on egg laying and adult emergence of Callosobruchus maculatus Fab. and on percentage of larval parasitism by Dinarmus vagabundus (Timberlake). Cowpea seeds treated with methanol and aqueous extract of Vitex, at these concentrations (0.5 and 1%), significantly reduced the number of eggs and emergence of F1 adults of C. maculatus. Both Vitex and Cassia extracts did not affect the percentage of parasitism by D. vagabundus on C. maculatus grubs.     

Identifying Elements of Patient-Centered Care in Underserved Populations: A Qualitative Study of Patient Perspectives

Patient-centered care is an important goal in the delivery of healthcare. However, many patients do not engage in preventive medical care. In this pilot study, we conducted twenty in depth, semi-structured qualitative interviews at the University of Illinois at Chicago Health Sciences campus in a four month time frame. Many patients were underserved and underinsured, and we wanted to understand their experiences in the healthcare system. Using content analysis, several themes emerged from the interview data. Participants discussed the need for empathy and rapport with their providers. They identified provider behaviors that fostered a positive clinical relationship, including step-by step explanations of procedures, attention to body language and clinic atmosphere, and appropriate time management. Participants identified cost as the most common barrier to engaging in preventive care and discussed children and social support as motivating factors. A long-term relationship with a provider was an important motivator for preventive care, suggesting that the therapeutic alliance was essential to many patients. Conversely, many participants discussed a sense of dehumanization in the healthcare system, reporting that their life circumstances were overlooked, or that they were judged based on insurance status or ethnicity. We discuss implications for provider training and healthcare delivery, including the importance of patient-centered medical homes.     

The Direct Actions of GABA, 2’-Methoxy-6-Methylflavone and General Anaesthetics at β3γ2L GABAA Receptors: Evidence for Receptors with Different Subunit Stoichiometries

2’-Methoxy-6-methylflavone (2’MeO6MF) is an anxiolytic flavonoid which has been shown to display GABA_A receptor (GABA_AR) β2/3-subunit selectivity, a pharmacological profile similar to that of the general anaesthetic etomidate. Electrophysiological studies suggest that the full agonist action of 2’MeO6MF at α2β3γ2L GABA_ARs may mediate the flavonoid’s in vivo effects. However, we found variations in the relative efficacy of 2’MeO6MF (2’MeO6MF-elicited current responses normalised to the maximal GABA response) at α2β3γ2L GABA_ARs due to the presence of mixed receptor populations. To understand which receptor subpopulation(s) underlie the variations observed, we conducted a systematic investigation of 2’MeO6MF activity at all receptor combinations that could theoretically form (α2, β3, γ2L, α2β3, α2γ2L, β3γ2L and α2β3γ2L) in Xenopus oocytes using the two-electrode voltage clamp technique. We found that 2’MeO6MF activated non-α-containing β3γ2L receptors. In an attempt to establish the optimal conditions to express a uniform population of these receptors, we found that varying the relative amounts of β3:γ2L subunit mRNAs resulted in differences in the level of constitutive activity, the GABA concentration-response relationships, and the relative efficacy of 2’MeO6MF activation. Like 2’MeO6MF, general anaesthetics such as etomidate and propofol also showed distinct levels of relative efficacy across different injection ratios. Based on these results, we infer that β3γ2L receptors may form with different subunit stoichiometries, resulting in the complex pharmacology observed across different injection ratios. Moreover, the discovery that GABA and etomidate have direct actions at the α-lacking β3γ2L receptors raises questions about the structural requirements for their respective binding sites at GABA_ARs.     

Opposite Expression of SPARC between the Liver and Pancreas in Streptozotocin-Induced Diabetic Rats

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that regulates several cellular events, including inflammation and tissue remodelling. In this study, we investigated the tissue-specific expression of SPARC in streptozotocin (STZ)-induced diabetes, and found that SPARC was significantly up-regulated in the liver while down-regulated in the pancreas of STZ-induced diabetic rats. Chronic inflammation occurred in the diabetic pancreas accompanied by up-regulation of CCAAT/enhancer-binding protein beta (C/EBPβ) and its targets (TNFα, Il6, CRP, and Fn1) as well as myeloperoxidase (Mpo) and C-X-C chemokine receptor type 2 (Cxcr2). Diabetic liver showed significant up-regulation of Tgfb1 as well as moderately less up-regulated TNFα and reduced Fn1, resulting in elevated fibrogenesis. PARP-1 was not up-regulated during CD95-mediated apoptosis, resulting in restoration of high ATP levels in the diabetic liver. On the contrary, CD95-dependent apoptosis was not observed in the diabetic pancreas due to up-regulation of PARP-1 and ATP depletion, resulting in necrosis. The cytoprotective machinery was damaged by pancreatic inflammation, whereas adequate antioxidant capacity indicates low oxidative stress in the diabetic liver. High and low cellular insulin content was found in the diabetic liver and pancreas, respectively. Furthermore, we identified six novel interacting partner proteins of SPARC by co-immunoprecipitation in the diabetic liver and pancreas, and their interactions with SPARC were predicted by bioinformatics tools. Taken together, opposite expression of SPARC in the diabetic liver and pancreas may be related to inflammation and immune cell infiltration, degrees of apoptosis and fibrosis, cytoprotective machinery, and cellular insulin levels.     

Determinants of Survival in Malignant Pleural Mesothelioma: A Surveillance,Epidemiology, and End Results (SEER) Study of 14,228 Patients

Introduction

Left untreated, malignant pleural mesothelioma (MPM) is associated with uniformly poor prognosis. Better survival has been reported with surgery-based multimodality therapy, but to date, no trial has demonstrated survival benefit of surgery over other therapies. We evaluated whether cancer-directed surgery influenced survival independently from other predictors in a large population-based dataset.

Methods

The SEER database was explored from 1973 to 2009 to identify all cases of pathologically-proven MPM. Age, sex, race, year of diagnosis, histology stage, cancer-directed surgery, radiation, and vital status were analyzed. The association between prognostic factors and survival was estimated using Cox regression and propensity matched analysis.

Results

There were 14,228 patients with pathologic diagnosis of MPM. On multivariable analysis, female gender, younger age, early stage, and treatment with surgery were independent predictors of longer survival. In comparison to no treatment, surgery alone was associated with significant improvement in survival [adjusted hazard ratio (adj HR) 0.64 (0.61–0.67)], but not radiation [adj HR 1.15 (1.08–1.23)]. Surgery and radiation combined had similar survival as surgery alone [adj HR 0.69 (0.64–0.76)]. Results were similar when cases diagnosed between 1973 and 1999 were compared to cases diagnosed between 2000 and 2009.

Conclusions

Despite developments in surgical and radiation techniques, the prognosis for MPM patients has not improved over the past 4 decades. Cancer-directed surgery is independently associated with better survival, suggesting that multimodal surgery-based therapy can benefit these patients. Further research in adjuvant treatment is necessary to improve prognosis in this challenging disease.     

Pyrethroid-Resistance and Presence of Two Knockdown Resistance (kdr) Mutations,F1534C and a Novel Mutation T1520I,in Indian Aedes aegypti

Background

Control of Aedes aegypti, the mosquito vector of dengue, chikungunya and yellow fever, is a challenging task. Pyrethroid insecticides have emerged as a preferred choice for vector control but are threatened by the emergence of resistance. The present study reports a focus of pyrethroid resistance and presence of two kdr mutations—F1534C and a novel mutation T1520I, in Ae. aegypti from Delhi, India.

Methodology/Principal Findings

Insecticide susceptibility status of adult-female Ae. aegypti against DDT (4%), deltamethrin (0.05%) and permethrin (0.75%) was determined using WHO''s standard insecticide susceptibility kit, which revealed resistance to DDT, deltamethrin and permethrin with corrected mortalities of 35%, 72% and 76% respectively. Mosquitoes were screened for the presence of kdr mutations including those reported earlier (I1011V/M, V1016G/I, F1534C, D1794Y and S989P), which revealed the presence of F1534C and a novel mutation T1520I. Highly specific PCR-RFLP assays were developed for genotyping of these two mutations. Genotyping using allele specific PCR and new PCR-RFLP assays revealed a high frequency of F1534C (0.41–0.79) and low frequency of novel mutation T1520I (0.13). The latter was observed to be tightly linked with F1534C and possibly serve as a compensatory mutation. A positive association of F1534C mutation with DDT and deltamethrin resistance in Ae. aegypti was established. However, F1534C-kdr did not show significant protection against permethrin.

Conclusions/Significance

The Aedes aegypti population of Delhi is resistant to DDT, deltamethrin and permethrin. Two kdr mutations, F1534C and a novel mutation T1520I, were identified in this population. This is the first report of kdr mutations being present in the Indian Ae. aegypti population. Highly specific PCR-RFLP assays were developed for discrimination of alleles at both kdr loci. A positive association of F1534C mutation with DDT and deltamethrin resistance was confirmed.     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.